Adenosine deaminase (ADA) is an enzyme of the purine catabolic pathway which is normally expressed in a tissue-specific manner. Deficiency of this enzyme is causally associated with a severe immunodeficiency disease, whereas increased levels of ADA are found in red cells of individuals with several disorders of erythropoiesis including Diamond- Blackfan syndrome. Adenosine deaminase activity has been found to be increased by 40- to 70- fold in red cells, but not in leukocytes, B lymphoblasts, or fibroblasts, from a kindred with an autosomal dominant form of hemolytic anemia. The elevated red cell ADA activity is associated with normal catalytic properties of the enzyme and normal structure of reticulocyte ADA mRNA. We wish to elucidate the molecular basis for this tissue-specific disorder of gene expression by achieving the following specific objectives: 1) determine whether the mutation responsible for this syndrome is cis- or trans- to the ADA gene. 2) examine the expression of ADA during normal maturation of red cell precursors using in situ hybridization and erythroid colony culture techniques; 3) examine the expression of ADA at the mRNA and transcriptional level during differentiation of human erythroleukemia nd/or leukemic T cell lines; 4), identify potential enhanced regions responsible for ADA regulation during differentiation using DNase I hypersensitive in the analysis; evaluate these regions as possible protein binding sites using gel retardation assays as in vivo regulators ADA expression in transient expression assays; 5) examine the promoter and potential enhanced regions for base alterations in individuals with the red cell ADA overproduction phenotype; 6) determine the functional significance of any such alterations using transient expression assays in erythroleukemia cell lines and retroviral transfer of the suspected regulatory element into murine hematopoietic stem cells; and 7) identify and characterize erythroid-specific trans-acting factors which may modulate ADA expression in erythroid precursors. These studies should enhance our understanding of the mechanisms governing normal gene expression during hematopoietic cell differentiation and may well lay the foundation for achieving more effective expression of ADA in immunodeficient patients as a result of gene transfer to hematopoietic stem cells.